Methods and apparatus for the selection and/or processing of particles, in particular for the selective and/or optimised lysis of cells

ABSTRACT

Methods and apparatus for the selection or processing of particles sensitive to the application of an external stimulus to rupture/lysis at least one selected particle or the fusion of first and second selected particles are disclosed herein. Particles are organized using a first field of force by selectively energizing electrodes of an array of selectable electrodes having dimensions comparable to or smaller than those of the particles. A first configuration of stresses is applied to the electrodes; and then a second configuration of stresses is applied to the electrodes, so as to create a second field of force, located substantially close to at least one selected particle to be lysated or to a pair of first and second particles to be fused and such as to produce the application of a stimulus suited to produce their lysis or fusion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S patent application Ser. No.12/296,952, which is a U.S National Phase of International PatentApplication No. PCT/IB2007000963, filed Apr. 12, 2007, which in turnclaims the priority benefit of Italian Patent Application No.TO2006A000273 filed Apr. 12, 2016, the entire respective disclosures ofwhich are incorporated herein by reference.

TECHNICAL FIELD

The present invention concerns methods and apparatus for the selectionand/or the processing of particles, in particular particles composed ofcells or including cells and/or cellular material, for example for theselective and/or optimised lysis of cells, and is applied principally inthe implementation of protocols with resolution on a single cell. Theterm “processing” of cells here and below means any type of operationthat can be carried out on a single particle or cell, or on a group ofthem.

STATE OF THE ART

The patent PCT/WO 00/69565 to G. Medoro describes an apparatus and amethod for the manipulation and identification/recognition of particlesmaking use of closed cages with dielectrophoretic potential and possibleintegrated sensors. The method described teaches how to control theposition of each particle independently of all the others in atwo-dimensional space. The force used to trap the particles suspended ina fluid medium is negative dielectrophoresis. The individual control ofthe manipulating operations is achieved by the programming of memoryelements and circuits associated with each element of an array ofelectrodes and sensors integrated in the same substratum. The deviceallows cells to be isolated, but requires that they be moved towards asecond microchamber, fluidly isolated from the first one. Moreover, nomethod is contemplated for transforming the cells.

The U.S. Pat. No. 6,294,063 to Becker et al. describes a method andapparatus for the manipulation of packages of solid, liquid or gaseousbiological material by means of a distribution of programmable forces.The patent also mentions the use of sensors. In this case too theisolation of the cells may take place only by moving the cellsphysically through the whole device.

A further force for the manipulation of particles is the force ofviscous friction generated by electro-hydrodynamic flows (EHD), such aselectrothermal flows (ETF) or AC electro-osmosis. In NG. Green, A. Ramosand H. Morgan, J. Phys. D: Appl. Phys. 33 (2000) the EHD are used toshift particles. For example PCT WO 2004/071668 A1 describes anapparatus for concentrating particles on electrodes, exploiting theabove-mentioned electro-hydrodynamic flows.

The Italian patent application BO2005A000481, Medoro et al., lists somemethods for manipulating particles with arrays of electrodes, and somemethods and apparatus for identifying them.

Instead the international patent application PCT/IT02/00524 describes amethod in which first biological entities may be transformed by beingput in contact with second biological entities (for example liposomescontaining DNA, or microbeads), where the first biological entities areimmobilised on a surface defined by a matrix of first electrodes whichmay be at least in part selectively activated and directed, placedfacing at least one second electrode, and are contacted with the secondbiological entities shifted by means of dielectrophoresis cages.

The patent application PCT IB 2006000636 in the name of the sameApplicant concerns a method and apparatus for the characterisationand/or count of particles by means of non uniform, time variable fieldsof force and integrated optical or impedenziometric sensors. The fieldsof force may have positive or negative dielectrophoresis,electrophoresis or electro-hydrodynamic movements, characterised by aset of stable points of equilibrium for the particles (solid, liquid orgaseous); the same method is suitable for the manipulation of drops(liquid particles) exploiting known effects such as Electrowetting ondielectric, with the aim of acting on the control of the position ofeach particle present in the specimen, so as to shift said particles ina deterministic or statistical way, to detect their presence with theintegrated optical or impedenziometric sensors, and/or to characterisetheir type, in order to count them or manipulate them in an efficientway.

In the Italian application in the name of the same Applicant, no.TO2006A000226 of 27 Mar. 2006, methods and apparatus are described forthe processing (for example washing, incubation, etc.) of particles inwhich the particles suspended in a first fluid are introduced underlaminar flow conditions into at least one first microchamber or firstregion of the same, in which a second fluid is introduced under laminarflow conditions into at least one second region of the microchamber orof a second microchamber, in such a way as not to mix with the firstfluid, and in which at least one field of force (F) acting on theparticles is activated in the microchamber(s), to provoke a shift of theparticles alone in a predetermined direction and to transfer the same insuspension into the second fluid; an apparatus is preferably usedincluding at least three microchambers arranged in sequence with eachother in one direction and each connected with the microchamberimmediately before it and after it with two orifices offset from eachother in a direction perpendicular to the direction of sequence of themicrochambers.

Recently, in the article A single cell electroporation chip, Lab on aChip, 2005, 5 (1), 38-43, Michelle Khine, Adrian Lau, CristianIonescu-Zanetti, Jeonggi Seo and Luke P. Lee, it has been described howto increase the permeability of the cellular membranes byelectroporation carried out on single cells; in this way, polarsubstances which could not otherwise permeate the plasmatic membrane(such as dyes, medicines, DNA, proteins, peptides and amino acids) canbe introduced into the cell.

The article Flow-through micro-electroporation chip for high efficiencysingle-cell genetic manipulation, Sensors and Actuators A: Physical.Volume 104, Issue 3, 15 May 2003, Pages 205-212, Yong Huang, BorisRubinsky describes in particular the genetic manipulation of individualcells, which is of great interest in fields such as biology andbiotechnologies, obtained by means of an electroporation chip whichmakes use of micro-fluid channels to manipulate single cells withprecision; as is known, electroporation is a technique that uses intenseelectrical fields to induce structural re-arrangements in the cellmembrane; pores are thus formed through the membrane when thetransmembrane potential exceeds the dielectric perforation voltage ofthe membrane (0.2-1.5V) allowing external substances to penetrate themembrane and reach the cytoplasm that it contains.

The electroporation of single cells is an interesting technique alsobecause it allows the study of the variations that occur in a cellpopulation cell by cell, and also the study of the intracellularchemistry, for example supplying specific phenotypes to individual cellsby activating or blocking the expression of specific and individualproteins. Using technology based on the use of matrices implemented onchips it is therefore possible to produce apparatus for HTS testing(high throughput screening), linked both to the expression of DNA andproteins and to chemical compounds (for example medicines) which aredirected towards specific cell targets (for example receptors).

The electroporation of single cells is also an advantageous technique incomparison with the protocols of electroporation in bulk that arenormally used, which require very high voltages (>103 V) and do notallow an efficacious control of the permeability of the individualcells, so that, for example, it is difficult to reclose the pores openedpreviously.

The attempts made so far to achieve the electroporation of single cellsrange from the use of carbon fibre microelectrodes (Lundqvist et al.,1998) to other techniques such as capillaries filled with electrolyte,micropipettes, and micromanufactured chips.

Micromanufactured devices are ideal both for isolating single cells andfor focussing the electric field.

Lastly, the article “Controlling cell destruction usingdielectrophoretic forces”, A. Menachery and R. Pethig, IEEProc.-Nanobiotechnol., Vol. 152, No. 4, August 2005, reports on a studyof the lysis of cells for different types of cells in castellated orpolynomial electrodes, and proposes the differential lysis of cells ofdifferent types present in a mixture (choosing frequencies andamplitudes such as to lysate one type, but save another).

However, as the electrodes are much bigger than the cells, it is notproposed to use this approach, and probably it is not possible to useit, to destroy single cells selectively, irrespective of their type. Infact, since the position with respect to relatively large electrodes(and consequently the intensity of the field to which they aresubjected) varies considerably, this method cannot work homogeneously onthe various cells.

Lysis is preferably induced using fields in an interval of frequenciesbetween the cross-over frequency (beyond which the cells pass fromnegative dielectrophoresis (nDEP) to positive (pDEP)), and lower thanthe frequency beyond which the potential of the membrane is attenuateddue to the exceeding of the membrane relaxation constant.

SUMMARY OF THE INVENTION

The aim of the present invention is to supply methods for operating onfluid samples containing particles, typically cells, for carrying outthe purification and/or isolation of single cells and/or thetransformation of one or more cells, which is without the limitationsand/or the inconveniences described for the prior art.

In particular an aim of the present invention is to act on the controlof the position of each particle present in the sample, in order toshift said particles in a deterministic way, to operate selectively oneach cell and/or perform in a more efficacious way operations such aslysis, fusion, etcetera.

Here and below, the terms “particles” or “particle” are used to indicatemicrometric or nanometric entities, natural or artificial, such ascells, subcellular components, viruses, liposomes, niosomes. The termcell will sometimes be used, but where not specified otherwise it mustbe understood as a non limiting example of the use of particles in thesense described more fully above.

The present invention therefore concerns the methods as specified in theclaims 1, 11, 12, 13, 15.

The invention also concerns an apparatus as specified in claim 17.

In particular, non uniform, time variable fields of force are used andintegrated optical sensors. The fields of force may have positive ornegative dielectrophoresis, electrophoresis or electro-hydrodynamicmovements, characterised by a set of stable points of equilibrium forthe particles.

In this way the limitations of the prior art are overcome by the presentinvention.

The implementation of the methods according to the invention allows theaccurate purification of a sample of cells even from contaminatingagents present in a low percentage. It also allows cells to betransformed efficaciously and selectively with the introduction ofgenetic material. Lastly it allows a few interesting cells to be rapidlyisolated from a heterogeneous sample.

Further characteristics and advantages of the invention will be clearfrom the following description of some of its non limiting embodiments,with reference to the figures in the attached drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates the steps of a first method accordingto the invention carried out in a manipulation apparatus illustrated insection in elevation;

FIG. 2 illustrates the steps of the method in FIG. 1 carried out withthe same apparatus as in FIG. 1, but illustrated in layout viewed fromabove;

FIG. 3 illustrates in the same view as FIG. 2 a possible variation ofthe method in FIGS. 1 and 2;

FIG. 4 illustrates the actuation of the method in FIG. 1 with aphotographic sequence;

FIG. 5 illustrates in the same view as FIG. 2 a further variation of themethod in FIG. 1;

FIG. 6 illustrates in the same view as FIG. 2 a further method of themanipulation of particles according to the present invention; and

FIGS. 7 to 9 illustrate different embodiments of an apparatus for aparticularly advantageous actuation of the methods of the invention.

DETAILED DESCRIPTION

The aim of the present invention is to carry out methods and apparatusfor the manipulation and/or separation and/or analysis of particles.

The methods of the invention are based (FIG. 1) on the use of a nonuniform field of force (F) with which to attract single particles orgroups of particles (CELL) towards positions of stable equilibrium(CAGE). This field may be, for example, a field of dielectrophoresis(DEP), negative (NDEP) or positive (PDEDP), or a field ofelectrohydrodynamic movements (EHD).

The processing carried out on the cells is based on the application oflocalised electric fields able to provoke the permanent rupture of thecellular membrane, or the fusion of two particles.

The method may also make use of integrated sensors, preferably of theoptical and/or impedenziometric type, for example in all those steps inwhich it is necessary to check the type of particles close to certainelectrodes. Alternatively, similar information may be available by meansof non integrated optical sensors, coupled to a microscope, which allowsthe examination of the contents of the microchamber in which the methodsof the invention are being carried out.

Generation of Forces

There are various methods for generating forces to shift particles,according to the prior art, by means of arrays of electrodes (EL),formed on a substratum. Typically, according to previous patents of thesame Applicant (FIG. 1), a cover (LID) is used, which may in turn be anelectrode, which delimits a microchamber, in which are the particles(CELL), typically suspended in a fluid composed of a liquid. In the caseof dielectrophoresis (DEP), the voltages applied are periodic in-phasevoltages (Vphip) indicated with the plus sign (+) and counterphasevoltages (Vphin) indicated with the minus sign (−). The term“counterphase voltages” means voltages offset by 180°. The fieldgenerates a force which acts on the particles of a region of space(CAGE), attracting them towards a point of equilibrium (PEQ). In thecase of negative DEP (NDEP), it is possible to produce closed forcecages, according to the prior art, if the cover (LID) is a conductiveelectrode; in this case the point of equilibrium (MPEQ) corresponds toeach electrode connected to Vphin (−) if the adjacent electrodes areconnected to the opposite phase Vphip (+) and if the cover (LID) isconnected to the phase Vphin (−). This point of equilibrium (MPEQ) isnormally at a distance in the liquid with respect to the electrodes, sothe particles (CELL) are in levitation, in a stationary state.

In the case of positive DEP (PDEP) the point of equilibrium (ZPEQ) isnormally at the surface on which the electrodes are realised, and theparticles (CELL) are in contact with it, in a stationary state. For thePDEP it is not necessary to have further electrodes in the cover,because the points of equilibrium of the PDEP correspond to the maximumsof the electric field. For the electro-hydrodynamic movements (EHD), theconfigurations of electrodes generate flows which push the particlestowards the minimum points of the flow.

For the sake of simplicity, below is considered purely as an example,and therefore without limitation for the purposes of the presentinvention, the use of closed cages with negative dielectrophoresis asthe activating force for the steps of particle movement in thedescription of the methods and apparatus (for which it is necessary touse a cover acting as an electrode) of the invention. To experts of thesector with ordinary abilities it is clear that it is possible togeneralise the methods and apparatus described below for the use ofdifferent activating forces, and different types of particles.

Method for the Selective Lysis of Particles

The particles to be lysated are positioned close to the gap between twoelectrodes by one of the above mentioned actuation forces, energisingthe electrodes with sinusoidal voltages of a first amplitude (MA) andfrequency (MF). The gap is preferably smaller than 10 μm, and typicallyaround 1-3 μm, so that a low voltage stimulus, compatible with thesupply voltage of an integrated circuit (e.g. 2.5, 3.3 or 5 V), isenough to determine a transmembrane potential sufficient to cause theirreversible rupture of the particle.

This stimulus is preferably composed of a train of sinusoidal impulsesof a second amplitude (ZA) and a second frequency (ZF).

Electric impulses are applied between the two selected electrodes so asto provoke the lysis of the cell.

FIG. 1 shows in section the evolution over time of the fields of forceand of the “patterns” (that is the complex of configurations of (+) or(−) state of the electrodes) of the voltages applied to the electrodes,according to a preferential embodiment of the invention. In FIG. 1(a),the cells (CELL) are in nDEP, suspended in the liquid in a first pointof equilibrium (MPEQ). In FIG. 1(b) the pattern of voltages applied tothe electrodes (EL) changes, so the frequency and optionally theamplitude of the voltages applied, as well as the force to which thecells are subjected, change to pDEP (FZAP). However, thanks to thechange of the pattern of voltages, only the cell to be lysated (CELLZ)is subjected to a significant force, so it is attracted towards a newpoint of stable equilibrium (ZPEQ). Near that point the electric fieldis maximum and the frequency is such that a sufficient transmembranepotential to lysate the cell is provoked.

FIG. 2 shows in layout the configurations of electrodes for the samesteps (a)-(d) as FIG. 1, where the pattern of electrodes in phase andcounterphase with the voltage applied to the lid is indicated by thecolour (in phase grey, in counterphase white).

This sequence of patterns is particularly favourable since, duringlysis, the other cells in neighbouring areas are subjected to an almostnull electric field, as both the cover and the electrodes are in phase.If the amplitude of the voltage applied to the cover is equal to theamplitude of the voltage applied to the electrodes, the field on thosecells is null.

Alternatively, the series of patterns shown in FIG. 3(a)-3(d) may beadopted. In this case, as is shown by the number of electrodes in phaseand counterphase represented by the colours white and grey, theadvantage lies in the need to reprogramme a smaller number of electrodeseach time, which may be advantageous if the process of writing thememory cells for the pattern of electrodes to be actuated is slow.Otherwise it is generally preferable to adopt the previous solution inFIG. 2.

With sensors integrated in the array of electrodes that delimit thebottom surface of the microchamber, for example of optical type, it iseasy to check when lysis has occurred, using the methods described inthe above-mentioned international patent application no. PCT IB2006000636 of the same Applicant, to see whether the cage correspondingto the lysated cell is still full or empty, or better to check for thepresence of debris resulting from lysis.

Substantially, with the methods described it is possible to select orprocess particles sensitive to the application of an external stimulususing a method comprising in general the step of producing, by applyingsaid external stimulus, the rupture or lysis of at least one selectedparticle; and wherein are also contemplated the steps of:

-   a) bringing the particles (CELL) close to electrodes (EL) of an    array of selectable electrodes having dimensions comparable to or    smaller than those of said particles, to which may be applied a    first pattern (PMAN) of tensions to organise optionally, if    necessary, said particles (CELL) by means of a first field of force    (FMAN), by selectively energising said electrodes (EL);-   b) applying to the electrodes a second pattern (PZAP) of voltages,    so as to create a second field of force (FZAP), located    substantially close to at least one selected particle to be lysated    (CELL) and such as to produce the application to said at least one    selected particle of a stimulus suited to produce its rupture or    lysis.

The particles are suspended in a chosen fluid, in case one wants to usethe passage from nDEP to pDEP as described previously to obtain lysis,so as to present a relatively low electric conductivity.

The first pattern (PMAN) of voltages for generating the first field offorce (FMAN) presents a first amplitude (MA) and a first frequency (MF);and the second pattern (PZAP) of voltages for generating the secondfield of force (FZAP) presents a second amplitude (ZA) and a secondfrequency (ZF), at least one of which is different from said firstamplitude (MA) and first frequency (MF). In this case, the stimulusapplied to obtain lysis consists of a force that can be applied to theat least one selected particle by the second field of force (FZAP) andboth the first and the second pattern of voltages are generated in AC(alternating current). In particular, the at least one selected particleis a biological entity with a lysable membrane, in the examplesdescribed a cell, and the stimulus applied consists of bringing thetransmembrane potential of the at least one selected particle to a valuesuch as to produce the rupture of the membrane.

According to a possible variation of the method of the invention, whichmay be considered illustrated in FIG. 2(b), the stimulus applied to theselected particle to lysate it consists vice versa of heating located inthe fluid in which is suspended the selected particle to be lysated.

According to this possible variation, particularly advantageous if theparticles are suspended in a fluid (liquid) presenting a high electricconductivity (for example physiological solution), the second pattern(PZAP) of voltages is such as to produce the selective heating by Jouleeffect of those selected electrodes in the array of electrodes withwhich the second field of force (FZAP) is generated, in FIG. 2(b) theelectrode shown in white, on which the whole current supplied to thedevice illustrated is practically concentrated.

In this case it is clear that at least the second pattern of voltagesmay be generated in either AC or DC (direct current).

Anyway, the methods described according to the invention include a stepof checking the lysis of the at least one selected particle, preferablycarried out by means of the already mentioned sensors integrated withthe array of electrodes, in a single chip.

Lastly, according to a further possible variation of the methodsdescribed, if one is interested in selectively recovering the debrisproduced by lysis, after the step b) described above, the followingsteps may be carried out:

-   c) applying said first pattern (PMAN) of voltages to the electrodes    again; and-   d) while step c) is in progress, producing a slow and controlled    shift of the fluid to recover a selected product of lysis of the at    least one selected particle.

In fact, as is well known to experts in the field, in the case ofactuating the movement of the particles by dielectrophoresis, the forcesacting on the particles due to the applied field are in proportion tothe cube of the radius of the particles, while the forces ofhydrodynamic viscous friction are in proportion only to the radius ofthe particles; therefore the smallest particles (the debris of lysis inthis specific case) may be carried along by a moderate flushing of thefluid in which the particles are suspended, while the largest particles(the non lysated cells) are kept in a stationary position (against theviscous flushing action) by the nDEP cages positioned in stationary modeand in which the cells are trapped.

The efficacy of the methods described, in particular of the methodaccording to the FIGS. 1 and 2, is shown in FIG. 4. Two Raji cellssuspended in an aqueous solution with Mannitol 280 mM (millimolar) andKCl 6.25 mM are organised, see FIG. 4(a), by the electric field (MF)applied (MA) in which the electrodes in the array have sinusoids with apeak-peak amplitude of 3.3 V, the conductive cover (LID) an amplitude of6.6V, all with frequency 50 kHz. The cells are taken onto the in-phaseelectrodes with the lid surrounded by electrodes of the opposite phase(offset by 180°).

After that, the pattern of the voltages applied to the electrodesvaries, putting into counterphase also the electrode on the cell atbottom right, which must be preserved. Although there is no cage, thecell remains in the same position, due to inertia. Instantaneously, theapplied electric field changes so as to produce positivedielectrophoresis (FZAP), bringing the frequency of the electric field(ZF) to 400 kHz, and the particle still present in the cage (CELLZ) goesinto a new point of stable equilibrium due to the force of positivedielectrophoresis, now generated by the field, which is on the gapbetween the electrodes, see FIG. 4(b). That region corresponds to themaximums of the electric field, which at that frequency are sufficientto provoke the lysis of the membrane, see FIG. 4(c).

It appears clear to experts in the sector with ordinary abilities thatthe electric field may be varied, in different ways, in particular also(or only) in amplitude, or the initial pattern of electrodes formanipulation and lysis may be chosen differently, for example as in FIG.5.

In this case, it starts from a nDEP pattern, FIG. 5(a), which positionsthe particles in a point of equilibrium (MPEQ) lying vertically to thepoint of equilibrium for the pDEP (ZPEQ), for the next pattern ofelectrodes. In this way the selected cell does not move from itsvertical line, and the lysis process can be accelerated because the celltakes less time to reach the area of maximum electric field in whichlysis takes place.

Method for Particle Fusion Assisted by Dielectrophoretic Manipulation

In this case, two particles are brought into contact in the same pointof stable equilibrium (MPEQ) by the force (F) generated by theelectrodes (EL). The stimulus that is applied next to the pair of cellsin contact is chosen with an amplitude and frequency such as to provokea fusion of the two cellular membranes into a single entity (FIG. 6).

With sensors integrated in the chip that holds the array of electrodes,for example of an optical type, it is then possible to check that fusionhas taken place without the need of an external microscope.

Applications of fusion include for example the generation of hybrids, ofboth eucariot cells and bacteria, or of plants.

For example, one could consider the possibility of using a similarmethod to reprogramme differentiated cells towards stem cells, forexample combining a differentiated cell with an enucleated stem cell.

So, according to this method, the fusion of first particles with secondparticles is produced, in which the first and second particles arebiological entities with a lysable membrane, for example cells ormicro-organisms, and in which the membrane of the particles is sensitiveto the application of an external stimulus, performing the steps of:

-   a) bringing said first and second particles (CELL) close to    electrodes (EL) of an array of selectable electrodes having    dimensions comparable to or smaller than those of said particles, to    which may be applied a first pattern (PMAN) of tensions to organise    optionally, if necessary, said first and second particles (CELL) by    means of a first field of force (FMAN), by selectively energising    said electrodes (EL);-   b) applying to the electrodes a second pattern (PZAP) of voltages,    so as to create a second field of force (FZAP), located    substantially close to at least one first and one second selected    particles to be fused together and such as to produce the    application to the same of a stimulus suited to produce the fusion    of the membranes of the first and second selected particles.    Method of Isolating Cells by Survival

A multiplicity of cells is flushed in suspension in the microchamberwith the array of electrodes. Using integrated sensors (for exampleoptical and/or impedenziometric) and/or external sensors (for example anoptical sensor coupled to a microscope, with or without fluorescence),the type or cell found in each point of equilibrium (PEQ) is identified.Stimuli for lysis are then applied selectively to all the cells that arenot interesting, preserving the vitality of the neighbouring interestingcells.

The sample is then flushed out, recovering the interesting live cellsand the lysate of non interesting cells.

According to the description, a method is therefore carried out forisolating interesting particles from a population of particles includingthe interesting particles, characterised in that it comprises thefollowing steps:

-   a) introducing the population of particles, suspended in a fluid,    into a microchamber, where the microchamber is provided with an    array of selectable electrodes;-   b) applying to said population of particles the method of selective    lysis described above to produce the selective lysis of all the    particles of the population except the interesting ones;-   c) recovering the interesting particles.

Step b), particularly in the case where the position of the interestingparticles with respect to the array of electrodes is not known a prioriand the interesting particles possess characteristics such as to besensitive only to a determined intensity and/or type of stimulus (alsonot known a priori), is applied simultaneously and/or repeatedly to allthe particles of the particle population by applying to said electrodesa plurality of voltage patterns suited to apply to said particlesstimuli of an intensity and/or type such as to produce, in this way, theselective lysis of all the particles in the particle population exceptthe interesting ones.

The different type may for example include the application of ACvoltages with determined frequencies, known to be ineffective in thelysis of the interesting particles, but to be efficacious for the lysisof the remaining particles. The different intensity may for example be agrowing intensity.

With respect to isolation based on moving the interesting cells into asecond microchamber for recovery, the method described above presentsthe following advantages:

1. Speed of Execution

The cells move slowly under the described forces (DEP, ETF, EHD) ofactuation, and the sorting based on moving the cells into a microchamberfor recovery is therefore relatively slow. Vice versa, the time tocomplete the sorting operation based on survival requires only one cellto have reached the nearest point of stable equilibrium (PEQ), and thetime of lysis of the cell (about one second), and it is not necessary tomove it through the whole selection microchamber.

2. A Cooling System is not Necessary

The cells to be eliminated can be lysated in series, working bysub-regions of the microchamber. In this way it is not necessary to havecooling systems, even for working on very large chips with relativelyconductive buffers, because the quantity of heat developed isproportional to the energised area, which may be made as small as onelikes.

3. The Chip May have Very Large Dimensions

As it is not necessary to energise the whole chip at the same time, theproblem of resistive drop on the tracks that carry the stimuli to thevarious electrodes is correspondingly reduced. Above all with relativelyconductive buffers, and with low pitch between electrodes, the resistivedrop on the tracks inside the chip and/or the drop on the conductivelayer of the lid (when NDEP cages are used for actuation) is notnegligible if the whole array of electrodes is energised. Energisingonly a part of the layer, the resistive load to be managed is limitedand the voltage drop on the tracks is decreased.

4. Operation Also with Cells that Cannot be Manipulated with the Fieldsof Force

If the electrode matrix is sufficiently dense (with dimensionscomparable to or smaller than the cells), so as to be able to performselective lysis on cells even without having selected them beforehand,the method of the invention can still be completed, in particular if thecells to be preserved, presenting different characteristics from the noninteresting cells, are sensitive to stimuli carried out at differentfrequencies of voltages applied to the electrodes.

5. Simplified Microfluidic Package

It is not necessary to have a double microchamber, but a singlecompartment is sufficient, and the recovery need not be selective, sothe contamination is not linked to the characteristic of the recoveryflow.

Any sensors integrated in the chip will be used to

-   1. determine the interesting particles.-   2. Check the lysis of the undesired cells.    Method of Ultra-purification of Cells

With enrichment techniques it is often easy to eliminate cells presentin proportions greater than the interesting cells by several degree ofmagnitude (for example with centrifugations in gradients of density orenrichment with magnetic balls, etc.). However it is sometimes difficultto eliminate the few contaminating cells that remain (for examplepresent in a proportion of 0.1-10%) to obtain a 100% pure sample. Thisis required for example if one wants to cultivate the interesting cellsbut these proliferate less rapidly than the non interesting ones, which,even if present in a low percentage, would then prevail downstream fromthe proliferation.

As in the selection method described previously, the cells areintroduced into the microchamber, the cells optionally line up in pointsof stable equilibrium near the electrodes (PEQ), and the contaminatingcells are eliminated by lysating them with the electrodes, obviouslyafter having identified them with suitable integrated or externalsensors, or on the basis of intrinsic differences such as the frequencyof the voltage applied to the electrodes which produces lysis.

In this case too, the integrated sensors, if they exist, can optionallybe used to

-   1. determine the interesting particles.-   2. Check the lysis of the undesired cells.

The 100% pure cells are then recovered, flushing out the sample.

Based on this aspect of the invention, a method is therefore actuatedfor the ultra-purification of interesting particles from contaminatingparticles, both contained in a population of particles, characterised inthat the isolation method described above is applied, where step b) isapplied only to the contaminating particles.

Method of Cell Analysis

The study at biomolecular level of DNA and/or of proteins in singlecells is increasingly interesting. According to an aspect of the presentinvention, a method is proposed for selecting and shifting selectedcells possibly from a multiplicity of cells from a first multiplicity ofpoints, and bringing them to a second multiplicity of points of analysisof micrometric dimensions, each point of analysis comprising at the mostone single cell. The cell is lysated and its content is analysed on thechip, for example according to known techniques such as PCR and/orcapillary electrophoresis on chip.

Based on the invention, a method is therefore supplied for the analysisof particles, characterised in that it comprises the steps of:

-   a) introducing said particles, suspended in a fluid, into a    microchamber;-   b) shifting said particles, selectively, each into a predetermined    point of analysis separate from said microchamber, but hydraulically    connected to it;-   c) applying to said particles present in said predetermined points    of analysis a method of selective lysis as described above;-   d) applying a defined analysis protocol on site to the respective    debris of the lysis of said particles;-   where said defined analysis protocol is chosen from the group    including: PCR, capillary electrophoresis on chip; combinations of    the previous ones.-   Cell Analysis Apparatus

To carry out the methods described, in particular the previous method ofanalysis, an apparatus is preferentially used as illustrated in theFIGS. 7, 8 and 9. The apparatus (FIG. 7) contains an array of electrodesas in the prior art, but it is characterised by a main microchamber(CHM) and by a multiplicity of secondary microchambers (CHJ). The mainmicrochamber may be filled with a sample comprising at least one cellthrough the respective inlets (IM1) and outlets (OM1). Each secondarymicrochamber (CHJ) is of larger dimensions, preferably substantiallycomparable to those of a cell, as shown in FIG. 7. Preferably eachsecondary microchamber is connected to the main microchamber through achannel (LCHG) with a configuration (length and/or shape) sufficient toprevent (avoid or at least limit) the dispersion of the sample bydiffusion and contamination towards other microchambers, in the timenecessary for the analysis. According to a possible variation (FIG. 8),there is a multiplicity of secondary microchambers for lysis (CHLJ)connected to a channel for capillary electrophoresis on chip, forexample with a cross junction (TJ). Alternatively a series of channelsfor capillary electrophoresis may be produced with a double T junction,according to the prior art. Optionally, at the end of the channel forcapillary electrophoresis there is an integrated sensor (SENS_J), of animpedenziometric and/or optical type, able to produce anelectropherogram based on the migration time of the compounds analysedfrom the junction (cross or double T) to the sensor itself. A furthervariation is illustrated in FIG. 9, where each microchamber of saidmultiplicity is connected to a capillary for electrophoresis (CAPJ)through a fluid outlet (OJ) of each secondary microchamber.

The invention claimed is:
 1. A method for selecting and processing offirst particles sensitive to the application of an external stimulus andlysing at least one selected first particle, comprising the steps of: a)providing an array of electrodes, each electrode having dimensionscomparable to or smaller than those of said first particles and eachelectrode being a selectable electrode; b) selectively energizing saidarray of electrodes to apply to all of said electrodes a first patternof voltages to generate a first field of force having first points ofstable equilibrium each positioned at a first position, wherein thefirst particles are attracted towards the first points of stableequilibrium and are situated at the first position; c) applying to allof said electrodes of said array a second pattern of voltages, differentfrom the first pattern of voltages, to generate a second field of forcehaving at least one second point of stable equilibrium positioned at asecond position in a gap between at least two adjacent electrodes inproximity of the at least one selected first particle to be lysed;wherein the at least one selected first particle to be lysed isattracted to the at least one second point of stable equilibrium andshifted to the second position, and wherein: i) said second field offorce applies, only at the second position at which at least one secondpoint of stable equilibrium is generated, a stimulus to lyse the atleast one selected first particle to be lysed and said second field offorce being designed such that at least one first particle that is notselected to be lysed remains at the first position; ii) said firstpattern of voltages and said second pattern of voltages are applied in atime sequence to the same said electrodes.
 2. The method according toclaim 1, wherein said stimulus consists of a localized heating of afluid in which said at least one selected first particle to be lysed isimmersed.
 3. The method according to claim 1, wherein said secondpattern of voltages is such as to produce the selective heating by Jouleeffect at the second position at which said second stable point ofstable equilibrium is generated.
 4. The method according to claim 1,wherein said second pattern of voltages is generated in either AC or DC.5. The method according to claim 1, wherein said first pattern ofvoltages for generating said first field of force presents a firstamplitude and a first frequency; and in that said second pattern ofvoltages for generating the second field of force presents a secondamplitude and a second frequency, at least one of which is differentfrom said first amplitude and first frequency.
 6. The method accordingto claim 5, wherein said stimulus consists of a force applicable to saidat least one selected first particle to be lysed by said second field offorce; said second pattern of voltages being generated in AC.
 7. Themethod according to claim 6, wherein said at least one selected firstparticle to be lysed is a biological entity with a lysable membrane inwhich said stimulus consists of bringing the transmembrane potential ofsaid at least one selected first particle to a value such as to producethe rupture of the membrane.
 8. The method according to claim 7, whereinsaid biological entity is a cell.
 9. The method according to claim 1,further comprising checking the lysis of said at least one selectedfirst particle to be lysed.
 10. The method of claim 9, wherein said stepof checking the lysis is carried out by a device with sensors integratedwith said array of electrodes in a single chip.
 11. The method accordingto claim 1, wherein said first particles are suspended in a fluid,further comprising choosing the fluid to present a low electricconductivity compared to an electrical conductivity of physiologicalfluids.
 12. A method for isolating interesting particles from apopulation of particles including said interesting particles andnon-interesting particles, comprising the steps of: a) introducing saidpopulation of particles suspended in a fluid into a microchamberequipped with an array of electrodes, each electrode having dimensionsidentical with or smaller than those of particles of the population ofparticles including said interesting particles and each electrode beingselectable; b) selectively energizing said electrodes of the array ofelectrodes to apply to all of said electrodes a first pattern ofvoltages to generate a first field of force having first points ofstable equilibrium each positioned a first position, wherein saidinteresting and non-interesting particles from the population ofparticles are attracted towards the first points of stable equilibriumand are situated at the first position; c) applying to all of saidelectrodes of said array of electrodes a second pattern of voltages,different from the first pattern of voltages, to generate a second fieldof force having second stable points of equilibrium each positioned at asecond position arranged in a gap between adjacent electrodes of saidarray of electrodes in proximity of the non-interesting particles,wherein the non-interesting particles of said population of particlesare attracted towards the second points of stable equilibrium andshifted to the second position and the interesting particles remain atthe first position; wherein i) said second field of force is appliessolely at the second points of stable equilibrium a stimulus suitable toselectively lyse the non-interesting particles ii) said first and secondpattern of voltages are applied in a time sequence to the same saidelectrodes; and the method further comprising the step of d) recoveringthe interesting particles.
 13. The method according to claim 12, whereinsaid step c) is applied simultaneously and/or repeatedly to thenon-interesting particles by applying to said electrodes a plurality ofvoltage patterns suited to apply to said non-interesting particlesstimuli of an intensity and/or type such as to produce the selectivelysis of the non-interesting particles.
 14. A method for theultra-purification of interesting particles from non-interestingparticles comprising contaminated particles, wherein the isolationmethod according to claim 12 is applied, where step c) is applied onlyto said contaminated particles.
 15. A method for the analysis ofparticles, comprising: a) introducing said particles, suspended in afluid, into a microchamber; b) shifting said particles, selectively,each into a predetermined point of analysis separate from saidmicrochamber, but hydraulically connected to it; c) applying to saidparticles present in said predetermined points of analysis a methodaccording to claim 1; d) applying a defined analysis protocol on site tothe respective debris of the lysis of said particles.
 16. The methodaccording to claim 15, where said defined analysis protocol is chosenfrom the group including: polymerase chain reaction, capillaryelectrophoresis on chip; or combinations of polymerase chain reactionand capillary electrophoresis on chip.
 17. A method for producing thefusion of first particles with second particles, in which the first andsecond particles are biological entities with a lysable membrane, and inwhich the membrane of said particles is sensitive to the application ofan external stimulus, comprising: a) providing an array of electrodes,each electrode having dimensions identical with or smaller than those ofsaid first and second particles and each electrode being selectable; b)selectively energizing said electrodes of said array of electrodes toapply to all of said electrodes a first pattern of voltages to generatea first field force having first points of stable equilibrium, whereinthe said first and second particles are attracted towards first pointsof stable equilibrium and situated at a first position in proximity ofsaid electrodes; and c) applying to all said electrodes of said array ofelectrodes a second pattern of voltages, different from the firstpattern to generate a second field of force having at least one secondpoint of stable equilibrium positioned at a second position in a gapbetween at least two adjacent electrodes of said array of electrodes inproximity to selected first and second particles; wherein the selectedfirst and second particles are attracted to the at least one secondpoint of stable equilibrium and shifted to the second position, andwherein i) said second field of force applies solely at the at least onesecond stable point of equilibrium a stimulus suitable to fuse membranesof the selected first and second particles to be fused together, ii)said first and second patterns of voltages are applied in a timesequence to the same said electrodes.
 18. A method for selection andprocessing of particles sensitive to an application of an externalstimulus, comprising: applying a first pattern of voltages to an arrayof electrodes to generate a first field of force having first points ofstable equilibrium, wherein the particles are attracted towards thefirst points of stable equilibrium and reside at a first position andthe particles comprise first particles to be ruptured or lysed andsecond particles; applying a second pattern of voltages to the array ofelectrodes to generate a second field of force having second points ofstable equilibrium each positioned at a second position between twoadjacent electrodes, wherein the two adjacent electrodes are disposed inproximity to the first particles such that only the first particles areattracted to the second points of stable equilibrium and shifted thesecond position while the second particles remain at the first position;wherein the second field of force generated by the second pattern ofvoltages is configured to also apply electric impulses between the twoadjacent electrodes to apply a stimulus to the first particles in thesecond position suitable to rupture or lyse the first particles, whereinthe first and second patterns of voltages are applied in a time sequenceto the same electrodes.